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Abstract

The Golgi apparatus is a highly dynamic organelle, which has many vital functions in cellular mechanisms like lipid metabolism, protein secretion, intracellular signaling, and regulation of cell division. First described in neurons, the ultrastructure and function of this complex network is still not well delineated. Recent studies have shown this secretory organelle also in dendrites of neurons, named Golgi outposts (GOs). To date, these GOs have been analyzed mainly by light microscopy while their ultrastructural appearance remained only poorly defined. To study the morphology of GOs in dendrites and to examine if GOs show a different ultrastructure to that of somatic Golgi apparatus, I established new applications of correlative light and electron microscopy in cultured neurons and brain slices, which combine fluorescence microscopy with the superior resolution of electron microscopy. Photo-oxidation-based approaches allow for the direct ultra-structural visualization of fluorescent markers in 3D, like genetically encoded green fluorescent proteins (GFPs). However, this challenging methodology had to be adapted to detect Golgi enzymes in neuronal cell cultures and brain tissue. Therefore, I first optimized the chemical fixation conditions for cultured hippocampal neurons to approximate the morphologic quality of high-pressure freeze fixation. Secondly, I enhanced sensitivity, reliability, and precision of photo-oxidation procedures on neurons, which allowed to analyse the 3D ultrastructure of GOs in dendrites of cultured neurons and mammalian brain slices. Correlative 3D microscopy showed that GOs are smaller in volume and have lower number of cisternae per stack compared to somatic Golgi apparatus of the same cell. Further, the number of peri-Golgi vesicles around GOs appears to be less. Additionally, while the stack polarity of somatic Golgi apparatus might switch along the ribbon, GOs have strictly unidirectional polarity. The technical achievements and enhanced sensitivity regarding correlative microscopy of GFPs in cultured neurons and brain slices allow for further ultrastructural investigations of GOs at different metabolic states to better understand their vital functions.